A new approach has been developed for studying water and solute equilibria in mitochondria and cells. The distribution of nonelectrolytes in mitochondria will be studied using radioactively labelled isotopes. A rigorous procedure has been developed for the analysis of data. Using these methods, it has proved possible to demonstrate the multiphase nature of matrix water; to quantitate the extent of 'normal' and 'abnormal' water; and to obtain estimates of matrix activity coefficients of nonelectrolyte probes. The objective of these studies is to test the new hypothesis that mitochondrial water exists in two distinct aqueous phases with different solution properties: 'Normal water' responds to changes in medium water activity (osmotic strength), while 'abnormal water' does not. 'Normal water' has solution properties for solute probes indistinguishable from bulk water. 'Abnormal water' virtually excludes some solutes while concentrating others. It is suggested that the abnormal phase in mitochondria may be structured by, and associated predominantly with, the inner membranes and cristae of mitochondria. These findings may provide new insights into the nature of the permeability barrier to transport across cell membranes. The results also suggest new ways of looking at old problems such as radiation protection, cryobiology, mechanisms of anesthetic action, mechanisms of drug-membrane interactions, the controversy over mechanisms of homeostasis and over the interpretation of n.m.r. data on cell water. If this model is correct, the true concentration of a solute does not, in general, equal the ratio of total solute to total water. The model can be applied to obtain true concentrations for the normal phase. This permits the more accurate determination of substrate concentrations within a phase, and of probes to determine the pH and electrical potential gradients in mitochondria.